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Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified Multiplex Ligation-dependent Probe Amplification approach.

Ohnesorg T, Eggers S, Leonhard WN, Sinclair AH, White SJ - BMC Genomics (2009)

Bottom Line: We were able to obtain reproducible results with as little as 5 x 10(4) cells per DNaseI treatment.Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method.This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Murdoch Children's Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, VIC, Australia.

ABSTRACT

Background: Mapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems.

Results: We have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA) approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 x 10(4) cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method.

Conclusion: This new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined) it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 10(5) cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions.

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Related in: MedlinePlus

Method overview. Schematic overview of the steps involved in DNaseI-MLPA, including approximate times required for each step when using four different cell preparations with three different DNaseI concentrations. Results can be obtained in less than 48 hours.
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Figure 1: Method overview. Schematic overview of the steps involved in DNaseI-MLPA, including approximate times required for each step when using four different cell preparations with three different DNaseI concentrations. Results can be obtained in less than 48 hours.

Mentions: Figure 1 outlines the protocol used in this study. To examine the general feasibility of our approach, we designed 11 probes to cover randomly chosen DNaseI hypersensitive sites in HeLa cells as published by the ENCODE consortium [21]. In addition, nine probes were designed in regions that showed no evidence of DNaseI hypersensitivity. To be able to cover larger genomic regions and to give greater flexibility in probe design we employed the recently developed extension MLPA [20]. All 20 probes were combined in a single mix, and could be differentiated from each other on the basis of length (the final product length range was 94-207 bp).


Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified Multiplex Ligation-dependent Probe Amplification approach.

Ohnesorg T, Eggers S, Leonhard WN, Sinclair AH, White SJ - BMC Genomics (2009)

Method overview. Schematic overview of the steps involved in DNaseI-MLPA, including approximate times required for each step when using four different cell preparations with three different DNaseI concentrations. Results can be obtained in less than 48 hours.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2748097&req=5

Figure 1: Method overview. Schematic overview of the steps involved in DNaseI-MLPA, including approximate times required for each step when using four different cell preparations with three different DNaseI concentrations. Results can be obtained in less than 48 hours.
Mentions: Figure 1 outlines the protocol used in this study. To examine the general feasibility of our approach, we designed 11 probes to cover randomly chosen DNaseI hypersensitive sites in HeLa cells as published by the ENCODE consortium [21]. In addition, nine probes were designed in regions that showed no evidence of DNaseI hypersensitivity. To be able to cover larger genomic regions and to give greater flexibility in probe design we employed the recently developed extension MLPA [20]. All 20 probes were combined in a single mix, and could be differentiated from each other on the basis of length (the final product length range was 94-207 bp).

Bottom Line: We were able to obtain reproducible results with as little as 5 x 10(4) cells per DNaseI treatment.Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method.This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Murdoch Children's Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, VIC, Australia.

ABSTRACT

Background: Mapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems.

Results: We have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA) approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 x 10(4) cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method.

Conclusion: This new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined) it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 10(5) cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions.

Show MeSH
Related in: MedlinePlus